(B-70) Effects of Hydrogel Substrate Mechanical Properties on Mesenchymal Stromal Cell Growth

Introduction:: Mesenchymal stromal cells (MSCs) have displayed high therapeutic potential for musculoskeletal injury treatments. MSCs release pro-healing factors and regulate activities of differentiated cells. To meet clinical dosing requirements, cultures often undergo multiple rounds of proliferation before use to obtain a large sample of cells from a small initial batch. For clinical translation, MSCs are expanded ex vivo; however, standard tissue culture plastic (TCP) substrates can reduce MSC potency and proliferation (1). Cell morphology may be used as an indicator of proliferation. In this work, novel hydrogel culture substrates with varying mechanical properties were developed to investigate MSC mechanosensitive signaling, cell morphology, and immunomodulatory protein secretion. We hypothesize that decreasing culture substrate stiffness will decrease mechanosensitive signaling, alter cell morphology, and increase protein secretion from MSCs.

Materials and Methods::

Hydrogels were fabricated across a varied stiffness range using 8-30 wt% poly(ethylene glycol) diacrylate polymer (PEGDA, 600Da) and functionalized with RGD for cell adhesion. Hydrogel Young’s moduli were then characterized via Bose TA3100 compression testing (n=5); stiffnesses ranged from 30kPa (8% PEGDA) to 2600kPa (30% PEGDA). Human bone marrow MSCs were seeded on 80, 800, and 2600kPa hydrogels and a TCP control at densities of 1000 cells/cm2 (n=5), cultured for 4 days, and stained for actin (phalloidin), nucleus (Hoescht), and mechanosensitive yes-associated protein (YAP). Cells were imaged via confocal microscopy. Morphology and stain intensity data was analyzed in a custom CellProfiler image analysis pipeline. Softer gel formulations were used for immunomodulatory protein secretion analysis; cells were seeded on 30, 420, and 800kPa hydrogels as well as a TCP control at densities of 1000 cells/cm2 (n=5). Protein secretion was measured by Luminex multiplex ELISA assay on conditioned media samples. Statistical significance between samples was measured via 1-way ANOVA and determined by p< 0.05.

Results, Conclusions, and Discussions::

YAP levels in the nuclear region significantly decreased with decreasing hydrogel stiffness; MSCs on the softest hydrogel (30kPa) had 43±4.6% lower nuclear YAP stain intensity than those on the stiffest gel (2600kPa), indicating higher mechanosensitive signaling on stiffer surfaces. This confirms that cells were internally reacting to changes in culture surface stiffness via altered mechanosensitive protein production. Further evidence that MSCs respond to substrate stiffness was found through morphological analysis.  MSCs grown on stiffer hydrogels (800-2600kPa) and TCP displayed generally rounded morphologies, having smaller areas, smaller perimeters, and less elongation; example cells can be seen in Figure 1a. MSCs on softer hydrogels (80-800kPa) displayed generally elongated morphologies, having larger areas, larger perimeters, and greater elongation than those on stiffer surfaces; this can be seen in Figure 1b.  Protein secretion generally decreased as surface stiffness increased; secretion of 9/11 measured proteins increased by on average ~3X on the softest hydrogel compared to other hydrogels (see Figure 2). The two proteins which were secreted at lower levels on the 30kPa gel, IFN⍺2, and MCP-1, are both pro-inflammatory factors. Thus, altering culture substrate stiffness may be a promising avenue to promote pro-regenerative protein secretion from MSCs while reducing inflammation.

Acknowledgements (Optional): :

References (Optional): : 1. Drela, K., et al. (2019). Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes. Stem cells international. 7012692.